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等离子体序列和两种等离子体系统的可用性,用于CRISPRi knockdownClostridioides difficile基因,没有抗生素选择

Michelle Chua^1,2, Daniel Erickson^1,2, James Collins^1,2,3

¹Department of Microbiology & Immunology, University of Louisville, Louisville, Kentucky, USA.

Microbiology resource announcements

|April 28, 2025

概括

创建了一个针对Clostridioides difficile的新型CRISPR干扰 (CRISPRi) 系统. 这种无抗生素的系统能够使基因稳定地被淘汰,通过准必需的walA基因来证明这一点.

科学领域:

微生物学微生物学
分子生物学分子生物学
基因工程是一种基因工程.

背景情况:

困难杆菌感染构成了重大的公共卫生挑战.
现有的C. difficile遗传工具通常依赖于抗生素选择,这可能是有问题的.
开发稳定,不依赖抗生素的系统对于研究C. difficile基因功能至关重要.

研究的目的:

开发和验证一种针对Clostridioides difficile.的新型双质粒CRISPR干扰 (CRISPRi) 系统.
建立一个不需要抗生素维护等离子体稳定的系统.
为了证明该系统在基因淘汰方面的有效性.

主要方法:

构建两个等离子体:pJAK184.tetR.PT5-3.dCas9用于四环素诱导的dCas9和pJC.15A.sgRNA.TA通过毒素-抗毒素系统和sgRNA表达保持稳定.
将dCas9表达盒集成到染色体中.
使用mCherry报告员进行sgRNA定制.
通过准必不可少的基因walA.A.来证明基因淘汰.

主要成果:

成功地建立了一个功能性的双等离子体CRISPRi系统用于Clostridioides difficile.
该系统确保了稳定的等离子体维持,而不需要选择抗生素.
实现了基本基因walA的有效淘汰,验证了系统的功能.

结论:

这种无抗生素的CRISPRi系统为Clostridioides difficile的基因操纵提供了宝贵的工具.
稳定维护机制简化了实验程序,并减少了与抗生素相关的潜在混因素.
该系统有助于研究C. difficile的基本基因和毒性因素,有助于开发新的治疗策略.

关键词:

克里斯普里是指克里斯普里.克洛斯特里迪奥伊德斯困难菌没有抗生素的维护维护.dCas9 的使用情况

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相关概念视频

Antibiotic Selection

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CRISPR and crRNAs

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...